PROJECT SUMMARY/ABSTRACT Plasmodium falciparum causes nearly 435,000 deaths annually worldwide. Victims of severe malaria are predominantly sub-Saharan children, who may present with symptoms of severe anemia or unarousable coma. The pathogenesis of severe malaria is poorly understood but mediated by the expression of adhesive variant surface antigens (VSAs) on infected red blood cells. These VSAs are involved in sequestration and rosetting, unique virulence factors that allow the parasite to evade host immune responses and prevent clearance in the spleen. In particular, ABO blood groups are critical for rosetting, which is the spontaneous binding of infected and uninfected erythrocytes together. Individuals with blood type A are particularly vulnerable to severe malaria, unlike those with blood type O who appear relatively protected against severe disease. A relatively unstudied family of VSAs, the repetitive interspersed family (RIFIN) proteins, have been recently found to be important in rosetting. RIFINs preferentially bind blood type A antigens, forming larger and tighter rosettes than in blood type O. RIFINs may mediate the association between ABO blood type and severe malaria susceptibility; these proteins also appear to be targets for protective immunity. Humoral immune responses against RIFINs have been correlated with asymptomatic infections. As such, we posit that a subset of RIFINs play a critical role in severe malaria pathogenesis and that individuals who lack immunity to this subset are the most susceptible to develop severe malaria. In a case-control study that began in Mali in 2014, our group collected blood and serum samples from severe malaria cases and matched controls with uncomplicated malaria. Here, we propose to identify and sequence RIFINs expressed in severe malaria using next-generation RNA-sequencing technologies. Using a custom protein microarray, we will then determine whether a lack of antibodies to these RIFIN proteins is associated with developing severe disease. In Aim 1, we will identify subgroups of RIFIN transcripts that are differentially expressed in subjects with severe malaria versus matched controls with uncomplicated malaria via RNA-Seq. We hypothesize that a subset of RIFIN-As will be predominantly expressed in severe malaria cases with blood type A, in contrast to expression of a heterogeneous group of RIFIN-As in other infections. In Aim 2, we will use the predominant RIFIN transcripts identified in Aim 1 and other parasite antigens to populate a custom protein microarray in order to define a subset of RIFIN proteins associated with increased vulnerability to severe malaria. We hypothesize that sera from children acutely ill with severe malaria will recognize fewer RIFIN proteins on a protein microarray and react to them less intensely than their convalescent sera and sera from matched controls with uncomplicated malaria, particularly for severe malaria cases with blood type A. The candidate will gain valuable expertise in bioinformatics analysis, particularly from microarrays and transcriptomic data from high-throughput sequencing platforms; use epidemiological techniques to conduct translational infectious disease research; and apply these skills to support effective public health interventions such as a vaccine.